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Variation among RNA transcript isoforms can be generated from alternative start and polyadenylation sites, and results in RNAs and proteins with different properties being generated from the same genomic sequence; here a new method termed transcript isoform sequencing is described in yeast, and the method allows a fuller exploration of transcriptome diversity across the compact yeast genome. Yeast transcription variants quantified The expression of eukaryotic genomes is a complicated matter, a long way from the old picture of a series of distinct protein-coding genes separated by less-important tracts of DNA. Lars Steinmetz and colleagues have used a novel technique termed TIF-Seq to demonstrate that the yeast genome containing around 6,000 protein-coding genes produces more than 1.88 million unique transcript isoforms (TIFs), defined as unique combinations of start (5′) and end (3′) RNA sequences. This work demonstrates that the complexity of overlapping transcript isoforms has been greatly underestimated previously. Transcript function is determined by sequence elements arranged on an individual RNA molecule. Variation in transcripts can affect messenger RNA stability, localization and translation 1 , or produce truncated proteins that differ in localization 2 or function 3 . Given the existence of overlapping, variable transcript isoforms, determining the functional impact of the transcriptome requires identification of full-length transcripts, rather than just the genomic regions that are transcribed 4 , 5 . Here, by jointly determining both transcript ends for millions of RNA molecules, we reveal an extensive layer of isoform diversity previously hidden among overlapping RNA molecules. Variation in transcript boundaries seems to be the rule rather than the exception, even within a single population of yeast cells. Over 26 major transcript isoforms per protein-coding gene were expressed in yeast. Hundreds of short coding RNAs and truncated versions of proteins are concomitantly encoded by alternative transcript isoforms, increasing protein diversity. In addition, approximately 70% of genes express alternative isoforms that vary in post-transcriptional regulatory elements, and tandem genes frequently produce overlapping or even bicistronic transcripts. This extensive transcript diversity is generated by a relatively simple eukaryotic genome with limited splicing, and within a genetically homogeneous population of cells. Our findings have implications for genome compaction, evolution and phenotypic diversity between single cells. These data also indicate that isoform diversity as well as RNA abundance should be considered when assessing the functional repertoire of genomes.

RNA polymerase II (RNAPII) transcription converts the DNA sequence of a single gene into multiple transcript isoforms that may carry alternative functions. Gene isoforms result from variable transcription start sites (TSSs) at the beginning and polyadenylation sites (PASs) at the end of transcripts. How alternative TSSs relate to variable PASs is poorly understood. Here, we identify both ends of RNA molecules in Arabidopsis thaliana by transcription isoform sequencing (TIF-seq) and report four transcript isoforms per expressed gene. While intragenic initiation represents a large source of regulated isoform diversity, we observe that ~14% of expressed genes generate relatively unstable short promoter-proximal RNAs (sppRNAs) from nascent transcript cleavage and polyadenylation shortly after initiation. The location of sppRNAs correlates with the position of promoter-proximal RNAPII stalling, indicating that large pools of promoter-stalled RNAPII may engage in transcriptional termination. We propose that promoter-proximal RNAPII stalling-linked to premature transcriptional termination may represent a checkpoint that governs plant gene expression. Gene isoforms result from variable transcription start sites (TSSs) and polyadenylation sites (PASs) at the end of transcripts. Here, the authors perform transcript isoform sequencing and find widespread promoter- proximal transcriptional termination in Arabidopsis , suggesting this may represent a checkpoint that regulates plant gene expression.

PoxtA and OptrA are ATP binding cassette (ABC) proteins of the F subtype (ABCF). They confer resistance to oxazolidinone and phenicol antibiotics, such as linezolid and chloramphenicol, which stall translating ribosomes when certain amino acids are present at a defined position in the nascent polypeptide chain. These proteins are often encoded on mobile genetic elements, facilitating their rapid spread amongst Gram-positive bacteria, and are thought to confer resistance by binding to the ribosome and dislodging the bound antibiotic. However, the mechanistic basis of this resistance remains unclear. Here we refine the PoxtA spectrum of action, demonstrate alleviation of linezolid-induced context-dependent translational stalling, and present cryo-electron microscopy structures of PoxtA in complex with the Enterococcus faecalis 70S ribosome. PoxtA perturbs the CCA-end of the P-site tRNA, causing it to shift by ∼4 Å out of the ribosome, corresponding to a register shift of approximately one amino acid for an attached nascent polypeptide chain. We postulate that the perturbation of the P-site tRNA by PoxtA thereby alters the conformation of the attached nascent chain to disrupt the drug binding site. PoxtA confers resistance to ribosome-targeting oxazolidinone (linezolid) and chloramphenicol antibiotics. Here, Crowe-McAuliffe et al. provide structural insights into how binding of PoxtA to the ribosome indirectly promotes drug dissociation.

5PSeq is a method for studying ribosome dynamics based on co-translational mRNA decay. Genome-wide sequencing and quantification of 5′ phosphorylated mRNA degradation products allows the positions of the last translating ribosomes to be determined. Co-translational mRNA degradation is a widespread process in which 5′–3′ exonucleolytic degradation follows the last translating ribosome, thus producing an in vivo ribosomal footprint that delimits the 5′ position of the mRNA molecule within the ribosome. To study this degradation process and ribosome dynamics, we developed 5PSeq, which is a method that profiles the genome-wide abundance of mRNA degradation intermediates by virtue of their 5′-phosphorylated (5′P) ends. The approach involves targeted ligation of an oligonucleotide to the 5′P end of mRNA degradation intermediates, followed by depletion of rRNA molecules, reverse transcription of 5′P mRNAs and Illumina high-throughput sequencing. 5PSeq can identify translational pauses at rare codons that are often masked when using alternative methods. This approach can be applied to previously extracted RNA samples, and it is straightforward and does not require polyribosome purification or in vitro RNA footprinting. The protocol we describe here can be applied to Saccharomyces cerevisiae and potentially to other eukaryotic organisms. Three days are required to generate 5PSeq libraries.

RT-LAMP detection of SARS-CoV-2 has been shown to be a valuable approach to scale up COVID-19 diagnostics and thus contribute to limiting the spread of the disease. Here we present the optimization of highly cost-effective in-house produced enzymes, and we benchmark their performance against commercial alternatives. We explore the compatibility between multiple DNA polymerases with high strand-displacement activity and thermostable reverse transcriptases required for RT-LAMP. We optimize reaction conditions and demonstrate their applicability using both synthetic RNA and clinical patient samples. Finally, we validate the optimized RT-LAMP assay for the detection of SARS-CoV-2 in unextracted heat-inactivated nasopharyngeal samples from 184 patients. We anticipate that optimized and affordable reagents for RT-LAMP will facilitate the expansion of SARS-CoV-2 testing globally, especially in sites and settings where the need for large scale testing cannot be met by commercial alternatives.

Progression of RNA polymerase II (RNAPII) transcription relies on the appropriately positioned activities of elongation factors. The resulting profile of factors and chromatin signatures along transcription units provides a \"positional information system\" for transcribing RNAPII. Here, we investigate a chromatin-based mechanism that suppresses intragenic initiation of RNAPII transcription. We demonstrate that RNAPII transcription across gene promoters represses their function in plants. This repression is characterized by reduced promoter-specific molecular signatures and increased molecular signatures associated with RNAPII elongation. The conserved FACT histone chaperone complex is required for this repression mechanism. Genome-wide Transcription Start Site (TSS) mapping reveals thousands of discrete intragenic TSS positions in fact mutants, including downstream promoters that initiate alternative transcript isoforms. We find that histone H3 lysine 4 mono-methylation (H3K4me1), an Arabidopsis RNAPII elongation signature, is enriched at FACT-repressed intragenic TSSs. Our analyses suggest that FACT is required to repress intragenic TSSs at positions that are in part characterized by elevated H3K4me1 levels. In sum, conserved and plant-specific chromatin features correlate with the co-transcriptional repression of intragenic TSSs. Our insights into TSS repression by RNAPII transcription promise to inform the regulation of alternative transcript isoforms and the characterization of gene regulation through the act of pervasive transcription across eukaryotic genomes.

Most mammalian promoters are inherently bidirectional, but transcription only elongates productively in one direction. Data presented in this paper demonstrate that at least part of the answer lies in the asymmetric distribution of polyadenylation-site sequences around human gene promoters causing termination of upstream antisense transcription. Active human promoters produce promoter-upstream transcripts (PROMPTs). Why these RNAs are coupled to decay, whereas their neighboring promoter-downstream mRNAs are not, is unknown. Here high-throughput sequencing demonstrates that PROMPTs generally initiate in the antisense direction closely upstream of the transcription start sites (TSSs) of their associated genes. PROMPT TSSs share features with mRNA-producing TSSs, including stalled RNA polymerase II (RNAPII) and the production of small TSS-associated RNAs. Notably, motif analyses around PROMPT 3′ ends reveal polyadenylation (pA)-like signals. Mutagenesis studies demonstrate that PROMPT pA signals are functional but linked to RNA degradation. Moreover, pA signals are under-represented in promoter-downstream versus promoter-upstream regions, thus allowing for more efficient RNAPII progress in the sense direction from gene promoters. We conclude that asymmetric sequence distribution around human gene promoters serves to provide a directional RNA output from an otherwise bidirectional transcription process.

Codon optimality is a major determinant of mRNA translation and degradation rates. However, whether and through which mechanisms its effects are regulated remains poorly understood. Here we show that codon optimality associates with up to 2-fold change in mRNA stability variations between human tissues, and that its effect is attenuated in tissues with high energy metabolism and amplifies with age. Mathematical modeling and perturbation data through oxygen deprivation and ATP synthesis inhibition reveal that cellular energy variations non-uniformly alter the effect of codon usage. This new mode of codon effect regulation, independent of tRNA regulation, provides a fundamental mechanistic link between cellular energy metabolism and eukaryotic gene expression. Synopsis Analysis of GTEx data and perturbation experiments in yeast show that cellular energy regulates the effect of optimal codon usage on mRNA stability. Codon optimality matters more in conditions of scarcer energy, such as tissues with low mitochondrial activity, older age, oxygen deprivation, or exposure to specific drugs. This effect can explain up to 2-fold variation in mRNA stability between human tissues and is reflected in the codon usage of tissue-specific cassette exons. ATP-inhibition experiments demonstrate a causal effect. While the underpinning mechanism is unknown, these observations support the independence of this mechanism from tRNA abundance regulation. Analysis of GTEx data and perturbation experiments in yeast show that cellular energy regulates the effect of optimal codon usage on mRNA stability.

The budding yeast Xrn1 protein shuttles between the nucleus, where it stimulates transcription, and the cytoplasm, where it executes the major cytoplasmic mRNA decay. In the cytoplasm, apart from catalyzing 5’→3’ decay onto non translated mRNAs, Xrn1 can follow the last translating ribosome to degrade the decapped mRNA template, a process known as “cotranslational mRNA decay”. We have previously observed that the import of Xrn1 to the nucleus is required for efficient cytoplasmic mRNA decay. Here by using an Xrn1 mutant that cannot enter the nucleus, but is otherwise functional in ribonuclease activity, we show that nuclear import is necessary for proper global cotranslational decay of mRNAs along coding regions and also affects degradation in the of 5’ region of a large group of mRNAs, which comprise about 20% of the transcriptome. Furthermore, a principal component analysis of the genomic datasets of this mutant and other Xrn1 mutants also shows that lack of a cytoplasmic 5’→3’ exoribonuclease is the primary cause of the physiological defects seen in a xrn1Δ mutant, but also suggests that Xrn1 import into the nucleus is necessary for its full in vivo functions.